Microorganisms such as filamentous fungi, yeast and algae produce a variety of lipids, including fatty acyls, glycerolipids, phospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids and prenol lipids. It is advantageous to extract some of these lipids from the microbial cells in which they are produced, and thus a variety of processes have been implemented.
One class of lipids commonly extracted from microbes is glycerolipids, including the fatty acid esters of glycerol (“triacylglycerols” or “TAGs”). TAGs are the primary storage unit for fatty acids, and thus may contain long chain polyunsaturated fatty acids (PUFAs), as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. There has been growing interest in including PUFAs, such as eicosapentaenoic acid [“EPA”; omega-3] and docosahexaenoic acid [“DHA”; omega-3], in pharmaceutical and dietary products. Means to efficiently and cost-effectively extract, refine and purify lipid compositions comprising PUFAs are therefore particularly desirable.
Many typical lipid isolation procedures involve disruption of the microbial cells (e.g., via mechanical, enzymatic or chemical means), followed by oil extraction using organic or green solvents. The disruption process releases the intracellular lipids from the microbial cells, which makes them readily accessible to the solvent during extraction. After extraction, the solvent is typically removed (e.g., by evaporation, for example by application of vacuum, change of temperature or pressure, etc.).
The resulting extracted oil is enriched in lipophilic components that accumulate in lipid bodies. In general, the major components of lipid bodies consist of TAGs, ergosterol esters, other sterol esters, free ergosterol and phospholipids. PUFAs present in lipid bodies are mainly as components of TAGs, diacylglycerols, monoacylglycerols and phospholipids, but can also be in the form of free fatty acids. The extracted oil may be subsequently refined to produce a highly purified TAG fraction enriched in PUFAs. Final specifications concerning the purified TAG fraction may be application-dependent, for example, depending on whether the oil is to be used as an additive or supplement (e.g., in food compositions, infant formulas, animal feeds, etc.), in cosmetic or pharmaceutical compositions, etc. Acceptable contaminant standards are either self-imposed (wherein a particular contaminant results in an undesirable property, e.g., haziness/cloudiness, odor) or determined by external nutrition councils (e.g., A Voluntary Monograph Of The Council for Responsible Nutrition (Washington, D.C.), March 2006, specifies the maximum acid, peroxide, anisidine, TOTOX, polychlorinated dibenzo-para-dioxin and polychlorinated dibenzofuran values for omega-3 EPA, omega-3 DHA and mixtures thereof).
U.S. Pat. No. 6,166,230 (GIST-Brocades) describes a process for treating a microbial oil comprising PUFAs (e.g., from Mortierella alpina) with a polar solvent to extract at least one sterol (e.g., desmosterol) that is soluble in the solvent and then separating at least some of the solvent containing the sterol from the oil, wherein the oil has a sterol content of less than 1.5%.
U.S. Pat. No. 7,695,626 (Martek) describes a process for recovering neutral lipids comprising PUFAs from a microbial biomass (e.g., Schizochytrium), said process comprising the steps of contacting the biomass with a nonpolar solvent to recover lipid in an extraction process, refining and/or bleaching and/or deodorizing the lipid composition, adding a polar solvent to the lipid composition, cooling the mixture to selectively precipitate at least one other compound (e.g., trisaturated glycerides, phosphorus-containing materials, wax esters, saturated fatty acid containing sterol esters, sterols, squalene, hydrocarbons) and then reducing the amount of this undesirable compound from the lipid composition.
Previous methods have not utilized techniques of short path distillation as an effective means to avoid exposing PUFAs, specifically highly unsaturated fatty acids, to high temperatures and reduce the amount of ergosterol (ergosta-5,7,22-trien-3β-ol; CAS Registry Number 57-87-4) contaminants from microbial oils.